Phenothiazine kinesin inhibitors

ABSTRACT

Phenothiazine derivatives of formula (I) are disclosed. The compounds are inhibitors of the mitotic kinesin KSP and are useful in the treatment of cellular proliferative diseases, such as cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S.S No. 60/263,092, filed Jan. 19, 2001, which is incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

[0002] This invention relates to phenothiazine derivatives which areinhibitors of the mitotic kinesin KSP and are useful in the treatment ofcellular proliferative diseases, for example cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation.

BACKGROUND OF THE INVENTION

[0003] Among the therapeutic agents used to treat cancer are the taxanesand vinca alkaloids, which act on microtubules. Microtubules are theprimary structural element of the mitotic spindle. The mitotic spindleis responsible for distribution of replicate copies of the genome toeach of the two daughter cells that result from cell division. It ispresumed that disruption of the mitotic spindle by these drugs resultsin inhibition of cancer cell division, and induction of cancer celldeath. However, microtubules form other types of cellular structures,including tracks for intracellular transport in nerve processes. Becausethese agents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

[0004] Improvements in the specificity of agents used to treat cancer isof considerable interest because of the therapeutic benefits which wouldbe realized if the side effects associated with the administration ofthese agents could be reduced. Traditionally, dramatic improvements inthe treatment of cancer are associated with identification oftherapeutic agents acting through novel mechanisms. Examples of thisinclude not only the taxanes, but also the camptothecin class oftopoisomerase I inhibitors. From both of these perspectives, mitotickinesins are attractive targets for new anti-cancer agents.

[0005] Mitotic kinesins are enzymes essential for assembly and functionof the mitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

[0006] Among the mitotic kinesins which have been identified is KSP. KSPbelongs to an evolutionarily conserved kinesin subfamily of plusend-directed microtubule motors that assemble into bipolar homotetramersconsisting of antiparallel homodimers. During mitosis KSP associateswith microtubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

[0007] Human KSP (also termed HsEg5) has been described (Blangy, et al.,Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, etal., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426), and a fragment of theKSP gene (TRIP5) has been described (Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number L40372). Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

[0008] Mitotic kinesins are attractive targets for the discovery anddevelopment of novel mitotic chemotherapeutics. Accordingly, it is anobject of the present invention to provide methods and compositionsuseful in the inhibition of KSP, a mitotic kinesin.

[0009] Phenothiazines have been known as psychopharmacologic agents formany years. Chlorpromazine, fluphenazine, perphenazine, trifluoperazine,promazine and thioridazine are typical examples. Inhibition of KSP byphenothiazines has not been described.

SUMMARY OF THE INVENTION

[0010] In accordance with the objects outlined above, the presentinvention provides compositions and methods that can be used to treatdiseases of proliferating cells. The compositions are KSP inhibitors,particularly human KSP inhibitors.

[0011] In one aspect, the invention relates to methods for treatingcellular proliferative diseases, for treating disorders associated withKSP kinesin activity, and for inhibiting KSP kinesin. The methods employcompounds of the formula:

[0012] wherein

[0013] R¹ is hydrogen, halogen or CF₃;

[0014] R² is chosen from hydrogen and lower alkyl;

[0015] R³ is hydrogen;

[0016] R⁴ and R⁵ are independently chosen from hydrogen, alkyl,substituted alkyl, alkylaryl, substituted alkylaryl, alkylheteroaryl andsubstituted alkylheteroaryl; or

[0017] any of R², R³ and R⁴ taken together with the intervening atomsform one or more five- to seven-membered rings, or a pharmaceuticallyacceptable salt thereof.

[0018] The ring may be substituted with one or more alkyl, aryl, alkoxy,halo, alkylaryl or substituted alkylaryl substituents. It is necessaryfor activity that the phenothiazine contain at least one five- toseven-membered ring in addition to the three rings of the phenothiazine.

[0019] Diseases and disorders that respond to therapy with compounds ofthe invention include cancer, hyperplasia, restenosis, cardiachypertrophy, immune disorders and inflammation.

[0020] In another aspect, the invention relates to compounds useful ininhibiting KSP kinesin. The compounds have the structures shown above.

[0021] In an additional aspect, the present invention provides methodsof screening for compounds that will bind to a KSP kinesin, for examplecompounds that will displace or compete with the binding of thecompositions of the invention. The methods comprise combining a labeledcompound of the invention, a KSP kinesin, and at least one candidateagent and determining the binding of the candidate bioactive agent tothe KSP kinesin.

[0022] In a further aspect, the invention provides methods of screeningfor modulators of KSP kinesin activity. The methods comprise combining acomposition of the invention, a KSP kinesin, and at least one candidateagent and determining the effect of the candidate bioactive agent on theKSP kinesin activity.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is directed to a class of novelphenothiazines that are modulators of mitotic kinesins. By inhibiting ormodulating mitotic kinesins, but not other kinesins (e.g., transportkinesins), specific inhibition of cellular proliferation isaccomplished. Thus, the present invention capitalizes on the findingthat perturbation of mitotic kinesin function causes malformation ordysfunction of mitotic spindles, frequently resulting in cell cyclearrest and cell death. The methods of inhibiting a human KSP kinesincomprise contacting an inhibitor of the invention with a KSP kinesin,particularly human KSP kinesins, including fragments and variants ofKSP. The inhibition can be of the ATP hydrolysis activity of the KSPkinesin and/or the mitotic spindle formation activity, such that themitotic spindles are disrupted. Meiotic spindles may also be disrupted.

[0024] An object of the present invention is to develop inhibitors andmodulators of mitotic kinesins, in particular KSP, for the treatment ofdisorders associated with cell proliferation. Traditionally, dramaticimprovements in the treatment of cancer, one type of cell proliferativedisorder, have been associated with identification of therapeutic agentsacting through novel mechanisms. Examples of this include not only thetaxane class of agents that appear to act on microtubule formation, butalso the camptothecin class of topoisomerase I inhibitors. Thecompositions and methods described herein can differ in theirselectivity and are preferably used to treat diseases of proliferatingcells, including, but not limited to cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.

[0025] Accordingly, the present invention relates to methods employingphenothiazines of the formula:

[0026] wherein

[0027] R¹ is hydrogen, halogen or CF₃;

[0028] R¹ is chosen from hydrogen and lower alkyl;

[0029] R³ is hydrogen;

[0030] R⁴ and R⁵ are independently chosen from hydrogen, alkyl,substituted alkyl, alkylaryl, substituted alkylaryl, alkylheteroaryl andsubstituted alkylheteroaryl; or

[0031] any of R², R³ and R⁴ taken together with the intervening atomsform one or more five- to seven-membered rings that may be optionallysubstituted with one or more alkyl, aryl, alkoxy, halo, alkylaryl orsubstituted alkylaryl substituents, or a pharmaceutically acceptablesalt thereof. It is necessary for activity that the phenothiazinecontain at least one five- to seven-membered ring in addition to thethree rings of the phenothiazine.

[0032] All of the compounds falling within the foregoing parent genusand its subgenera are useful as kinesin inhibitors, but not all thecompounds are novel. In particular, certain known species fall withinthe genus in which R⁴ and R⁵ have the full breadth of operativesubstituents, although no utility in inhibiting kinesin has beensuggested for these species. Any narrowing of the claims or specificexceptions that might be added to these claims reflect applicants'intent to avoid claiming subject matter that, while functionally part ofthe inventive concept, is not patentable to them for reasons havingnothing to do with the scope of their invention. In particular, thenovel compounds that are the subject of the claims are described by theformula:

[0033] wherein R¹, R² and R³ are as defined above;

[0034] R^(4a) is chosen from hydrogen and lower alkyl; and

[0035] R^(5a) is chosen from alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or

[0036] any of R², R³ and R^(4a) taken together with the interveningatoms form one or more five-to seven-membered rings, which may beoptionally substituted with one or more alkyl, aryl, alkoxy, halo,alkylaryl or substituted alkylaryl substituents, or a pharmaceuticallyacceptable salt thereof.

[0037] Preferred compounds of the methods and compositions are those inwhich R³ is hydrogen and R² and R^(4a) form a five- to seven-memberedring. Such compounds include phenothiazines of formula

[0038] In most preferred compounds R^(5a) is benzyl or substitutedbenzyl.

[0039] Other preferred compounds of the methods and compositions arethose in which R² and R³ are hydrogen and R^(5a) is alkylaryl orsubstituted alkylaryl, particularly those in which R^(5a) is benzyl orsubstituted benzyl.

[0040] Definitions

[0041] Alkyl is intended to include linear, branched, or cyclichydrocarbon structures and combinations thereof. Lower alkyl refers toalkyl groups of from 1 to 5 carbon atoms. Examples of lower alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and thelike. Preferred alkyl groups are those of C₂₀ or below. More preferredalkyl groups are those of C₁₃ or below. Cycloalkyl is a subset of alkyland includes cyclic hydrocarbon groups of from 3 to 13 carbon atoms.Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl,norbornyl, adamantyl and the like. In this application, alkyl refers toalkanyl, alkenyl and alkynyl residues; it is intended to includecyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene refersto the same residues as alkyl, but having two points of attachment.Examples of alkylene include ethylene (—CH₂CH₂—), propylene(—CH₂CH₂CH₂—), dimethylpropylene (—CH₂C(CH₃)₂CH₂—) andcyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When an alkyl residue having aspecific number of carbons is named, all geometric isomers having thatnumber of carbons are intended to be encompassed; thus, for example,“butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl;“propyl” includes n-propyl and isopropyl.

[0042] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms ofa straight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy andthe like. Lower-alkoxy refers to groups containing one to four carbons.

[0043] Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. Lower-acyl refers to groups containingone to four carbons.

[0044] Aryl and heteroaryl mean a 5- or 6-membered aromatic orheteroaromatic ring containing 0-3 heteroatoms selected from O, N, or S;a bicyclic 9- or 10-membered aromatic or heteroaromatic ring systemcontaining 0-3 heteroatoms selected from O, N, or S; or a tricyclic 13-or 14-membered aromatic or heteroaromatic ring system containing 0-3heteroatoms selected from O, N, or S. The aromatic 6- to 14-memberedcarbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin,and fluorene and the 5- to 10-membered aromatic heterocyclic ringsinclude, e.g., imidazole, pyridine, indole, thiophene, benzopyranone,thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline,pyrimidine, pyrazine, tetrazole and pyrazole.

[0045] Alkylaryl refers to a residue in which an aryl moiety is attachedto the parent structure via an alkyl residue. Examples are benzyl,phenethyl, phenylvinyl, phenylallyl and the like. Alkylheteroaryl refersto a residue in which a heteroaryl moiety is attached to the parentstructure via an alkyl residue. Examples include furanylmethyl,pyridinylmethyl, pyrimidinylethyl and the like.

[0046] Heterocycle means a cycloalkyl or aryl residue in which one tofour of the carbons is replaced by a heteroatom such as oxygen, nitrogenor sulfur. Examples of heterocycles that fall within the scope of theinvention include imidazoline, pyrrolidine, pyrazole, pyrrole, indole,quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran,benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl,when occurring as a substituent), tetrazole, morpholine, thiazole,pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline,isoxazole, dioxane, tetrahydrofuran and the like. Examples ofsubstituted heterocyclyl include 4-methyl-1-piperazinyl and4-benzyl-1-piperidinyl.

[0047] Substituted alkyl, aryl and heteroaryl or heterocyclyl refer toalkyl, aryl, heteroaryl or heterocyclyl wherein H atoms are replacedwith alkyl, halogen, hydroxy, alkoxy, alkylenedioxy (e.g.methylenedioxy) fluoroalkyl, carboxy (—COOH), carboalkoxy (i.e. acyloxyRCOO—), carboxyalkyl (—COOR), carboxamido, sulfonamidoalkyl,sulfonamidoaryl, aminocarbonyl, benzyloxycarbonylamino (CBZ-amino),cyano, carbonyl, nitro, dialkylamino, alkylamino, amino, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, arylthio, arylsulfinyl,arylsulfonyl, amidino, phenyl, benzyl, heteroaryl, heterocyclyl,phenoxy, benzyloxy, or heteroaryloxy. For the purposes of the presentinvention, substituted alkyl also includes oxaalkyl residues, i.e. alkylresidues in which one or more carbons has been replaced by oxygen.

[0048] Halogen refers to fluorine, chlorine, bromine or iodine.Fluorine, chlorine and bromine are preferred.

[0049] Many of the compounds described herein contain one or moreasymmetric centers (e.g. the carbons to which R² and R³ are attached)and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)— or (S)—. The present invention is meant toinclude all such possible isomers, including racemic mixtures, opticallypure forms and intermediate mixtures. Optically active (R)- and(S)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques. When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

[0050] When desired, the R- and S-isomers may be resolved by methodsknown to those skilled in the art, for example by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallisation; via formation of diastereoisomericderivatives which may be separated, for example, by crystallisation,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticoxidation or reduction, followed by separation of the modified andunmodified enantiomers; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support, such as silica witha bound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

[0051] The graphic representations of racemic, ambiscalemic and scalemicor enantiomerically pure compounds used herein are taken from Maehr J.Chem. Ed. 62, 114-120 (1985): solid and broken wedges are used to denotethe absolute configuration of a chiral element; wavy lines indicatedisavowal of any stereochemical implication which the bond it representscould generate; solid and broken bold lines are geometric descriptorsindicating the relative configuration shown but denoting racemiccharacter; and wedge outlines and dotted or broken lines denoteenantiomerically pure compounds of indeterminate absolute configuration.

[0052] In some embodiments, two R groups may be joined to form a ringstructure. Again, the ring structure may contain heteroatoms and may besubstituted with one or more substituents.

[0053] The compositions of the invention are synthesized as outlinedbelow, utilizing techniques well known in the art. Once made, thecompositions of the invention find use in a variety of applications. Aswill be appreciated by those in the art, mitosis may be altered in avariety of ways; that is, one can affect mitosis either by increasing ordecreasing the activity of a component in the mitotic pathway. Stateddifferently, mitosis may be affected (e.g., disrupted) by disturbingequilibrium, either by inhibiting or activating certain components.Similar approaches may be used to alter meiosis.

[0054] In a preferred embodiment, the compositions of the invention areused to modulate mitotic spindle formation, thus causing prolonged cellcycle arrest in mitosis. By “modulate” herein is meant altering mitoticspindle formation, including increasing and decreasing spindleformation. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

[0055] The compositions of the invention are useful to bind to and/ormodulate the activity of a mitotic kinesin, KSP. In a preferredembodiment, the KSP is human KSP, although KSP kinesins from otherorganisms may also be used. In this context, modulate means eitherincreasing or decreasing spindle pole separation, causing malformation,i.e., splaying, of mitotic spindle poles, or otherwise causingmorphological perturbation of the mitotic spindle. Also included withinthe definition of KSP for these purposes are variants and/or fragmentsof KSP. See U.S. Patent Application “Methods of Screening for Modulatorsof Cell Proliferation and Methods of Diagnosing Cell ProliferationStates”, filed Oct. 27, 1999 (U.S. Ser. No. 09/428,156), herebyincorporated by reference in its entirety. In addition, other mitotickinesins may be used in the present invention. However, the compositionsof the invention have been shown to have specificity for KSP.

[0056] For assay of activity, generally either KSP or a compoundaccording to the invention is non-diffusably bound to an insolublesupport having isolated sample receiving areas (e.g., a microtiterplate, an array, etc.). The insoluble support may be made of anycomposition to which the compositions can be bound, is readily separatedfrom soluble material, and is otherwise compatible with the overallmethod of screening. The surface of such supports may be solid or porousand of any convenient shape. Examples of suitable insoluble supportsinclude microtiter plates, arrays, membranes and beads. These aretypically made of glass, plastic (e.g., polystyrene), polysaccharides,nylon or nitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. Theparticular manner of binding of the composition is not crucial so longas it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. Following bindingof the protein or agent, excess unbound material is removed by washing.The sample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

[0057] The antimitotic agents of the invention may be used on their ownto modulate the activity of a mitotic kinesin, particularly KSP. In thisembodiment, the mitotic agents of the invention are combined with KSPand the activity of KSP is assayed. Kinesin activity is known in the artand includes one or more kinesin activities. Kinesin activities includethe ability to affect ATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes; such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

[0058] Methods of performing motility assays are well known to those ofskill in the art. (See e.g., Hall, et al. (1996), Biophys. J., 71:3467-3476, Turner et al., 1996, Anal. Biochem. 242 (1):20-5; Gittes etal., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J. Exp.Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53;Winkelmann et al., 1995, Biophys. J. 68: 72S.)

[0059] Methods known in the art for determining ATPase hydrolysisactivity also can be used. Preferably, solution based assays areutilized. U.S. application Ser. No. 09/314,464, filed May 18, 1999,hereby incorporated by reference in its entirety, describes such assays.Alternatively, conventional methods are used. For example, P_(i) releasefrom kinesin can be quantified. In one preferred embodiment, the ATPasehydrolysis activity assay utilizes 0.3 M PCA (perchloric acid) andmalachite green reagent (8.27 mM sodium molybdate II, 0.33 mM malachitegreen oxalate, and 0.8 mM Triton X-1 00). To perform the assay, 10 μL ofreaction is quenched in 90 μL of cold 0.3 M PCA. Phosphate standards areused so data can be converted to mM inorganic phosphate released. Whenall reactions and standards have been quenched in PCA, 100 μL ofmalachite green reagent is added to the relevant wells in e.g., amicrotiter plate. The mixture is developed for 10-15 minutes and theplate is read at an absorbance of 650 nm. If phosphate standards wereused, absorbance readings can be converted to mM P_(i) and plotted overtime. Additionally, ATPase assays known in the art include theluciferase assay.

[0060] ATPase activity of kinesin motor domains also can be used tomonitor the effects of modulating agents. In one embodiment ATPaseassays of kinesin are performed in the absence of microtubules. Inanother embodiment, the ATPase assays are performed in the presence ofmicrotubules. Different types of modulating agents can be detected inthe above assays. In a preferred embodiment, the effect of a modulatingagent is independent of the concentration of microtubules and ATP. Inanother embodiment, the effect of the agents on kinesin ATPase can bedecreased by increasing the concentrations of ATP, microtubules or both.In yet another embodiment, the effect of the modulating agent isincreased by increasing concentrations of ATP, microtubules or both.

[0061] Agents that modulate the biochemical activity of KSP in vitro maythen be screened in vivo. Methods for such agents in vivo include assaysof cell cycle distribution, cell viability, or the presence, morphology,activity, distribution, or amount of mitotic spindles. Methods formonitoring cell cycle distribution of a cell population, for example, byflow cytometry, are well known to those skilled in the art, as aremethods for determining cell viability. See for example, U.S. PatentApplication “Methods of Screening for Modulators of Cell Proliferationand Methods of Diagnosing Cell Proliferation States,” filed Oct. 22,1999, Ser. No. 09/428,156, hereby incorporated by reference in itsentirety.

[0062] In addition to the assays described above, microscopic methodsfor monitoring spindle formation and malformation are well known tothose of skill in the art (see, e.g., Whitehead and Rattner (1998), J.Cell Sci. 111:2551-61; Galgio et al, (1996) J. Cell biol., 135:399-414).

[0063] The compositions of the invention inhibit the KSP kinesin. Onemeasure of inhibition is IC₅₀, defined as the concentration of thecomposition at which the activity of KSP is decreased by fifty percent.Preferred compositions have IC₅₀'s of less than about 1 mM, withpreferred embodiments having IC₅₀'s of less than about 100 μM, with morepreferred embodiments having IC₅₀'s of less than about 10 μM, withparticularly preferred embodiments having IC₅₀'s of less than about 1μM, and especially preferred embodiments having IC₅₀'s of less thanabout 500 nM. Measurement of IC₅₀ is done using an ATPase assay.

[0064] Another measure of inhibition is K_(i). For compounds with IC₅₀'sless than 1 μM, the K_(i) or K_(d) is defined as the dissociation rateconstant for the interaction of the phenothiazine with KSP. Preferredcompounds have K_(i)'s of less than about 100 μM, with preferredembodiments having K_(i)'s of less than about 10 μM, and particularlypreferred embodiments having K_(i)'s of less than about 1 μM andespecially preferred embodiments having K_(i)'s of less than about 500nM.

[0065] Another measure of inhibition is GI₅₀, defined as theconcentration of the compound that results in a decrease in the rate ofcell growth by fifty percent. Preferred compounds have GI₅₀'s of lessthan about 1 mM. The level of preferability of embodiments is a functionof their GI₅₀: those having GI₅₀'s of less than about 20 μM are morepreferred; those having GI₅₀'s of 10 μM more so; those having GI₅₀ ofless than about 1 μM more so; those having GI₅₀'s of 500 nM more so.Measurement of GI₅₀ is done using a cell proliferation assay.

[0066] The compositions of the invention are used to treat cellularproliferation diseases. Disease states which can be treated by themethods and compositions provided herein include, but are not limitedto, cancer (further discussed below), autoimmune disease, arthritis,graft rejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper or hypo proliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly; as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

[0067] The compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc.More particularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymornma,germinoma (pinealoma), glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia (acuteand chronic), acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignantlymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

[0068] Accordingly, the compositions of the invention are administeredto cells. By “administered” herein is meant administration of atherapeutically effective dose of the mitotic agents of the invention toa cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantcells in which mitosis or meiosis can be altered.

[0069] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals, and otherorganisms. Thus the methods are applicable to both human therapy andveterinary applications. In the preferred embodiment the patient is amammal, and in the most preferred embodiment the patient is human.

[0070] Mitotic agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agents may be administered alone orin combination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

[0071] In a preferred embodiment, the pharmaceutical compositions are ina water soluble form, such as pharmaceutically acceptable salts, whichis meant to include both acid and base addition salts. “Pharmaceuticallyacceptable acid addition salt” refers to those salts that retain thebiological effectiveness of the free bases and that are not biologicallyor otherwise undesirable, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

[0072] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

[0073] The administration of the mitotic agents of the present inventioncan be done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the anti-mitotic agents maybe directly applied as a solution or spray.

[0074] To employ the compounds of the invention in a method of screeningfor compounds that bind to KSP kinesin, the KSP is bound to a support,and a compound of the invention (which is a mitotic agent) is added tothe assay. Alternatively, the compound of the invention is bound to thesupport and KSP is added. Classes of compounds among which novel bindingagents may be sought include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of particular interest are screening assays for candidate agentsthat have a low toxicity for human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, functional assays (phosphorylation assays, etc.) andthe like.

[0075] The determination of the binding of the mitotic agent to KSP maybe done in a number of ways. In a preferred embodiment, the mitoticagent (the compound of the invention) is labeled, for example, with afluorescent or radioactive moiety and binding determined directly. Forexample, this may be done by attaching all or a portion of KSP to asolid support, adding a labeled mitotic agent (for example a compound ofthe invention in which at least one atom has been replaced by adetectable isotope), washing off excess reagent, and determining whetherthe amount of the label is that present on the solid support. Variousblocking and washing steps may be utilized as is known in the art.

[0076] By “labeled” herein is meant that the compound is either directlyor indirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

[0077] In some embodiments, only one of the components is labeled. Forexample, the kinesin proteins may be labeled at tyrosine positions using¹²⁵I, or with fluorophores. Alternatively, more than one component maybe labeled with different labels; using ¹²⁵I for the proteins, forexample, and a fluorophor for the mitotic agents.

[0078] The compounds of the invention may also be used as competitors toscreen for additional drug candidates. “Candidate bioactive agent” or“drug candidate” or grammatical equivalents as used herein describe anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., to be tested for bioactivity. Theymay be capable of directly or indirectly altering the cellularproliferation phenotype or the expression of a cellular proliferationsequence, including both nucleic acid sequences and protein sequences.In other cases, alteration of cellular proliferation protein bindingand/or activity is screened. Screens of this sort may be performedeither in the presence or absence of microtubules. In the case whereprotein binding or activity is screened, preferred embodiments excludemolecules already known to bind to that particular protein, for example,polymer structures such as microtubules, and energy sources such as ATP.Preferred embodiments of assays herein include candidate agents which donot bind the cellular proliferation protein in its endogenous nativestate termed herein as “exogenous” agents. In another preferredembodiment, exogenous agents further exclude antibodies to KSP.

[0079] Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

[0080] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

[0081] Competitive screening assays may be done by combining KSP and adrug candidate in a first sample. A second sample comprises a mitoticagent, KSP and a drug candidate. This may be performed in either thepresence or absence of microtubules. The binding of the drug candidateis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to KSP and potentially modulating its activity. That is, if thebinding of the drug candidate is different in the second sample relativeto the first sample, the drug candidate is capable of binding to KSP.

[0082] In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

[0083] In one embodiment, the candidate agent is labeled. Either thecandidate agent, or the competitor, or both, is added first to KSP for atime sufficient to allow binding, if present. Incubations may beperformed at any temperature which facilitates optimal activity,typically between 4 and 40° C.

[0084] Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

[0085] In a preferred embodiment, the competitor is added first,followed by the candidate agent. Displacement of the competitor is anindication the candidate agent is binding to KSP and thus is capable ofbinding to, and potentially modulating, the activity of KSP. In thisembodiment, either component can be labeled. Thus, for example, if thecompetitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidateagent is labeled, the presence of the label on the support indicatesdisplacement.

[0086] In an alternative embodiment, the candidate agent is added first,with incubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

[0087] It may be of value to identify the binding site of KSP. This canbe done in a variety of ways. In one embodiment, once KSP has beenidentified as binding to the mitotic agent, KSP is fragmented ormodified and the assays repeated to identify the necessary componentsfor binding.

[0088] Modulation is tested by screening for candidate agents capable ofmodulating the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

[0089] Alternatively, differential screening may be used to identifydrug candidates that bind to the native KSP, but cannot bind to modifiedKSP.

[0090] Positive controls and negative controls may be used in theassays. Preferably all control and test samples are performed in atleast triplicate to obtain statistically significant results. Incubationof all samples is for a time sufficient for the binding of the agent tothe protein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

[0091] A variety of other reagents may be included in the screeningassays. These include reagents like salts, neutral proteins, e.g.,albumin, detergents, etc which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The mixture of components may be added in anyorder that provides for the requisite binding.

[0092] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference intheir entirety.

EXAMPLES

[0093] Abbreviations and Definitions

[0094] The following abbreviations and terms have the indicated meaningsthroughout:

[0095] Ac=acetyl

[0096] BNB=4-bromomethyl-3-nitrobenzoic acid

[0097] Boc=t-butyloxy carbonyl

[0098] Bu=butyl

[0099] c-=cyclo

[0100] CBZ=carbobenzoxy=benzyloxycarbonyl

[0101] DBU=diazabicyclo[5.4.0]undec-7-ene

[0102] DCM=dichloromethane=methylene chloride=CH₂Cl₂

[0103] DCE=dichloroethane

[0104] DEAD=diethyl azodicarboxylate

[0105] DIC=diisopropylcarbodiimide

[0106] DIEA=N,N-diisopropylethylamine

[0107] DMAP=4-N,N-dimethylaminopyridine

[0108] DMF=N,N-dimethylformamide

[0109] DMSO=dimethyl sulfoxide

[0110] DVB=1,4-divinylbenzene

[0111] EDCI=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0112] EEDQ=2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

[0113] Et=ethyl

[0114] Fmoc=9-fluorenylmethoxycarbonyl

[0115] GC=gas chromatography

[0116] HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

[0117] HMDS=hexamethyldisilazane

[0118] HOAc=acetic acid

[0119] HOBt=hydroxybenzotriazole

[0120] Me=methyl

[0121] mesyl=methanesulfonyl

[0122] MTBE=methyl t-butyl ether

[0123] NMO=N-methylmorpholine oxide

[0124] PEG=polyethylene glycol

[0125] Ph=phenyl

[0126] PhOH=phenol

[0127] PfP=pentafluorophenol

[0128] PPTS=pyridinium p-toluenesulfonate

[0129] Py=pyridine

[0130] PyBroP=bromo-tris-pyrrolidino-phosphonium hexafluorophosphate

[0131] rt=room temperature

[0132] sat=d=saturated

[0133] s-=secondary

[0134] t-=tertiary

[0135] TBDMS=t-butyldimethylsilyl

[0136] TES=triethylsilane

[0137] TFA=trifluoroacetic acid

[0138] THF=tetrahydrofuran

[0139] TMOF=trimethyl orthoformate

[0140] TMS=trimethylsilyl

[0141] tosyl=p-toluenesulfonyl

[0142] Trt=triphenylmethyl

[0143] Synthesis of Compounds

[0144] The syntheses of several prototypical phenothiazines are shownbelow. Other phenothiazines are made in analogous fashion:

[0145] Phenothiazine Synthesis-Procedure A

[0146] Phenothiazine Synthesis-Procedure B

[0147] Phenothiazine Procedure A

[0148] Synthesis ofBenzyl-[2-(2-chloro-phenothiazin-10-yl)-ethyl]-methylamine fumarate.

[0149] Synthesis of (2-chlorophenothiazin-10-yl)acetic Acid.

[0150] 2-Chloro-10H-phenothiazine (10.0 g, 42.8 mmol) was dissolved inTHF (100 mL) and DMSO (10 mL). Sodium hydride (1.0 g, 43.4 mmol) wasadded, and the mixture was heated to reflux until gas evolution ceased.Ethyl bromoacetate (10 g, 59.9 mmol) was added slowly via syringe. Themixture was heated at reflux for 12 h. Sodium hydride (1.0 g, 43.4 mmol)and ethyl bromoacetate (5 g, 29.9 mmol) were added, and the mixture washeated an additional 12 h. The mixture was cooled to room temperatureand carefully diluted with water followed by ethyl acetate. The layerswere separated, and the aqueous layer extracted with ethyl acetate. Theorganic layers were combined, dried (Na₂SO₄), filtered, and concentratedto give a dark purple oil (11.8 g). The oil was dissolved in methanol(300 mL), and aqueous sodium hydroxide (6 N, 22 mL, 132 mmol) was added.The mixture was heated to reflux for 4 h. The mixture was cooled to roomtemperature, and the solvent removed under vacuum. The residue wasdissolved in water and was extracted with diethyl ether (ether layersdiscarded). The layers were separated, and the pH of the aqueous layerwas made acidic with concentrated hydrochloric acid. The water layer wasextracted with methylene chloride. The organic layers were combined,dried (Na₂SO₄), filtered, and concentrated to give(2-chlorophenothiazin-10-yl)acetic acid (7.38 g, 56%) as a brown solid.

[0151] Synthesis ofN-Benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide

[0152] (2-Chlorophenothiazin-10-yl)acetic acid (480 mg, 1.65 mmol) wasdissolved in THF (10 mL). To this solution,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.58 g,8.25 mmol), 1-hydroxybenzotriazole (220 mg, 1.65 mmol), and N-methylbenzyl amine (2.0 mL, 16.5 mmol) were added. The mixture was stirred atroom temperature overnight and was then diluted with water and methylenechloride. The aqueous layer was made basic with sodium hydroxide (1N),and the layers were separated. The aqueous layer was extracted withmethylene chloride. The organic layers were combined, washed with waterand brine, dried (Na₂SO₄), filtered, and concentrated to provide amixture of N-methyl benzyl amine and product (3.8 g). The residue waspurified with a plug of SiO₂. The product was recrystallized from hexaneand ethyl acetate to provideN-benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide as a whitesolid (258 mg, 40%).

[0153] Synthesis of Benzyl-[2-(2-chlorophenothiazin-10-yl)-ethyl]methylAmine Fumarate.

[0154] N-Benzyl-2-(2-chlorophenothiazin-10-yl)-N-methylacetamide (140mg, 0.355 mmol) was dissolved in THF (5 mL). A solution of borane.THFcomplex in THF (1.0 M, 5 mL, 5 mmol) was added, and the solution heatedto reflux for 4 h. The mixture was carefully diluted with saturated HClin methanol and stirred for 30 min. The solvent was removed undervacuum, and the residue dissolved in ethyl acetate and aqueous sodiumhydroxide (1 N). The layers were separated and the aqueous layerextracted with ethyl acetate. The organic layers were combined, dried(Na₂SO₄), filtered, and concentrated to providebenzyl-[2-(2-chlorophenothiazin-10-yl)-ethyl]methyl amine as a whitesolid (160 mg). The crude product was purified by SiO₂ chromatography toprovide pure product (50 mg, 34%) and 100 mg of impure product. The pureproduct was dissolved in acetone and a solution of fumaric acid (0.8 mL,0.02 g/mL in methanol) was added. The solvents were removed undervacuum, and the residue slurried in chloroform and filtered to providethe sub-titled compound (47 mg) as an off-white solid.

[0155] Phenothiazine Procedure B

[0156] Synthesis of2-Chloro-10-(1-pyridin-3-ylmethylpiperidin-3-yl)-10H-phenothiazineHydrochloride.

[0157] Synthesis of 2,4-dichloro-2′-nitrodiphenyl Thioether.

[0158] 2,4-Dichlorobenzenethiol (16.1 g, 89.9 mmol) was dissolved inethanol (200 mL) and was heated to reflux. A solution of sodium acetate(11.1 g, 135 mmol) dissolved in 100 mL ethanol was added, followed by asolution of 1-iodo-2-nitrobenzene (33.6 g, 135 mmol) in ethanol (100 mL,added in small portions over 5 min). The mixture was heated at refluxfor 12 h. The mixture was cooled to room temperature and concentratedunder vacuum. The residue was dissolved in water and methylene chloride,and the aqueous layer was diluted with saturated sodium bicarbonate. Thelayers were separated, and the aqueous layer was extracted withmethylene chloride. The organic layers were combined, dried (Na₂SO₄),filtered, and concentrated to provide a solid. The residue was washedwith 30% ethanol in water (300 mL) and collected by vacuum filtration.The filter cake was rinsed with 30% ethanol in water. The solid wastreated with methanol (100 mL), and the product collected by filtrationto provide 2,4-dichloro-2′-nitrodiphenyl thioether (25.6 g, 94%) as ayellow solid.

[0159] Synthesis of 2,4-dichloro-2′-aminodiphenyl Thioether

[0160] 2,4-Dichloro-2′-nitrodiphenyl thioether (22.4 g, 75 mmol) wasdissolved in ethyl acetate (125 mL) at 40° C. Adams catalyst (PtO₂, 2.5g) was added, and hydrogen was vigorously bubbled through the solutionfor 1 h. The mixture was stirred overnight under a static atmosphere ofhydrogen. The mixture was cooled to room temperature and filteredthrough a bed of cellulose. The solvent was removed under vacuum toprovide 2,4-dichloro-2′-aminodiphenyl thioether (18.1 g, 89%) as ayellow oil.

[0161] Synthesis of 3-Hydroxypiperidine-1-carboxylic Acid Tert-butylEster

[0162] 3-Hydroxypiperidine (10.0 g, 98.9 mmol) was dissolved inmethylene chloride (100 mL) and cooled to 0° C. Di-tert-butyldicarbonate (27.3 g, 125 mmol) was added at once (copious gas evolutionwas observed). The mixture was stirred for 12 h at room temperature. Thesolvent was removed under vacuum to provide3-hydroxypiperidine-1-carboxylic acid tert-butyl ester (19.8 g, 100%).

[0163] Synthesis of 3-Oxopiperidine-1-carboxylic Acid Tert-butyl Ester.

[0164] A mixture of DMSO (7.8 mL, 109.3 mmol) and methylene chloride(100 mL) was cooled to −78° C. Oxalyl chloride (4.8 mL, 54.7 mmol) wasadded to this solution via syringe. The mixture was stirred at −78° C.for 30 min. A solution of 3-hydroxypiperidine-1-carboxylic acidtert-butyl ester (10.0 g, 49.7 mmol) in methylene chloride (30 mL) wasadded dropwise to this solution (temperature remained below −70° C.).The mixture was stirred for 30 min. Triethylamine (28 mL, 200 mmol) wasadded dropwise over 20 min. The mixture was stirred at −78° C. for 1 hand was allowed to warm to room temperature and stirred for 45 min. Themixture was diluted with water, and the layers separated. The organiclayer was washed with water, dried Na₂SO₄), filtered, and concentratedto give 3-oxopiperidine-1-carboxylic acid tert-butyl ester (10.2 g) as ayellow, brown liquid.

[0165] Synthesis of3-[2-(2,4-Dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicAcid Tert-butyl Ester.

[0166] 2,4-Dichloro-2′-aminodiphenyl thioether (10.3 g, 38.1 mmol) wasdissolved in dichloroethane (120 mL). To this solution, a solution of3-oxopiperidine-1-carboxylic acid tert-butyl ester (14.0 g, 70.3 mmol)in dichloroethane (20 mL) and solid sodium triacetoxyborohydride (14.5g, 68.7 mmol) were slowly added. Acetic acid (5.4 mL, 94 mmol) was addedslowly via syringe, and the mixture stirred for 48 h. The mixture wascarefully diluted with water, and the pH was adjusted to approximately 9with aqueous sodium hydroxide (1 N). The mixture was diluted with ethylacetate, and the layers were separated. The aqueous layer was extractedwith ethyl acetate. The organic layers were combined, dried (Na₂SO₄),filtered, and concentrated to provide3-[2-(2,4-dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicacid tert-butyl ester as a brown oil. The crude product was purified bySiO₂ chromatography resulting in pure product (8.4 g, 49%) as a lightyellow oil.

[0167] Synthesis of3-(2-Chlorophenothiazin-10-yl)piperidine-1-carboxylic Acid Tert-butylEster.

[0168]3-[2-(2,4-Dichlorophenylsulfanyl)phenylamino]piperidine-1-carboxylicacid tert-butyl ester (8.4 g, 18.5 mmol) was dissolved in DMF (120 mL)and vigorously degassed with N₂ for 20 min. Cesium carbonate (27.2 g,83.5 mmol), copper(I) iodide (5.28 g, 27.8 mmol), and copper powder (8.4g, 1321 mmol) were added to the solution. The suspension was stirredvigorously and heated to 155-156° C. for 12 hours (N₂ was continuouslybubbled through the mixture). The mixture was cooled to room temperatureand diluted with ethyl acetate. The solids were removed by vacuumfiltration and were rinsed with ethyl acetate. The filtrate wasconcentrated under vacuum to remove 90% of the volatiles. The residuewas purified by SiO₂ chromatography to provide3-(2-chlorophenothiazin-10-yl) piperidine-1-carboxylic acid tert-butylester (4.1 g, 53%) as a white solid.

[0169] Synthesis of 2-Chloro-10-piperidin-3-yl-10H-phenothiazineHydrochloride

[0170] 3-(2-Chlorophenothiazin-10-yl) piperidine-1-carboxylic acidtert-butyl ester (4.1 g, 9.83 mmol) was dissolved in diethyl ether (200mL), and the solution cooled to O ° C. A solution of HCl in ethylacetate (˜4 M, 30 mL) was slowly added. The mixture was allowed to warnto room temperature and stirred for 8 h. The solvent was removed undervacuum. The residue treated with HCl in ethyl acetate (˜4 M, 75 mL) andstirred overnight. The solvent was removed under vacuum to provide2-chloro-10-piperidin-3-yl-10H-phenothiazine (3.98 g, 100%) as anoff-white solid.

[0171] Synthesis of2-Chloro-10-(1-pyridin-3-yl-methylpiperidin-3-yl)-10H-phenothiazineHydrochloride.

[0172] Using methods substantially equivalent to those described in thesynthesis of3-[2-(2,4-dichloro-phenylsulfanyl)-phenylamino]-piperidine-1-carboxylicacid tert-butyl ester,

[0173]2-chloro-10-(1-pyridin-3-yl-methylpiperidin-3-yl)-10H-phenothiazine wasprepared by treatment of 2-chloro-10-piperidin-3-yl-10H-phenothiazine(240 mg, 0.757 mmol) and 3-pyridinecarboxaldehyde (81 mg, 0.757 mmol)with sodium triacetoxyborohydride (224 mg, 1.06 mmol) to provide aftertreatment with ethereal HCl, the sub-titled compound (136 mg, 41%).

[0174] Induction of Mitotic Arrest in Cell Populations Treated with aPhenothiazine KSP Inhibitor

[0175] FACS analysis to determine cell cycle stage by measuring DNAcontent was performed as follows. Skov-3 cells (human ovarian cancer)were split 1:10 for plating in 10 cm dishes and grown to subconfluencewith RPMI 1640 medium containing 5% fetal bovine serum (FBS). The cellswere then treated with either 10 nM paclitaxel, the test compound or0.25% DMSO (vehicle for compounds) for 24 hours. Cells were then rinsedoff the plates with PBS containing 5 mM EDTA, pelleted, washed once inPBS containing 1% FCS, and then fixed overnight in 85% ethanol at 4° C.Before analysis, the cells were pelleted, washed once, and stained in asolution of 10 g propidium iodide and 250%1 g of ribonuclease (RNAse) Aper milliliter at 37° C. for half an hour. Flow cytometry analysis wasperformed on a Becton-Dickinson FACScan, and data from 10,000 cells persample was analyzed with Modfit software.

[0176] The phenothiazine compounds, as well as the known anti-mitoticagent paclitaxel, caused a shift in the population of cells from a G0/G1cell cycle stage (2n DNA content) to a G2/M cell cycle stage (4n DNAcontent). Other compounds of this class were found to have similareffects.

[0177] Monopolar Spindle Formation Following Application of aPhenothiazine KSP Inhibitor

[0178] To determine the nature of the G2/M accumulation, human tumorcell lines Skov-3 (ovarian), HeLa (cervical), and A549 (lung) wereplated in 96-well plates at densities of 4,000 cells per well (SKOV-3 &HeLa) or 8,000 cells per well (A549), allowed to adhere for 24 hours,and treated with various concentrations of the phenothiazine compoundsfor 24 hours. Cells were fixed in 4% formaldehyde and stained withantitubulin antibodies (subsequently recognized usingfluorescently-labeled secondary antibody) and Hoechst dye (which stainsDNA).

[0179] Visual inspection revealed that the phenothiazine compoundscaused cell cycle arrest in the prometaphase stage of mitosis. DNA wascondensed and spindle formation had initiated, but arrested cellsuniformly displayed monopolar spindles, indicating that there was aninhibition of spindle pole body separation. Microinjection of anti-KSPantibodies also causes mitotic arrest with arrested cells displayingmonopolar spindles.

[0180] Inhibition of Cellular Proliferation in Tumor Cell Lines Treatedwith Phenothiazine KSP Inhibitors.

[0181] Cells were plated in 96-well plates at densities from 1000-2500cells/well of a 96-well plate (depending on the cell line) and allowedto adhere/grow for 24 hours. They were then treated with variousconcentrations of drug for 48 hours. The time at which compounds areadded is considered T₀. A tetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (I.S>U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) was usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours was compared to the number of viable cells atthe time of drug addition, allowing for calculation of growthinhibition.

[0182] The growth over 48 hours of cells in control wells that had beentreated with vehicle only (0.25% DMSO) is considered 100% growth and thegrowth of cells in wells with compounds is compared to this.Phenothiazine KSP inhibitors inhibited cell proliferation in human tumorcell lines of the following tumor types: lung (NCI-H460, A549), breast(MDA-MB-231, MCF-7, MCF-7/ADR-RES), colon (HT29, HCT15), ovarian(SKOV-3, OVCAR-3), leukemia (HL-60(TB), K-562), central nervous system(SF-268), renal (A498), osteosarcoma (U2-OS), and cervical (HeLa). Inaddition, a mouse tumor line (B16, melanoma) was also growth-inhibitedin the presence of the phenothiazine compounds.

[0183] A Gi₅₀ was calculated by plotting the concentration of compoundin μM vs the percentage of cell growth of cell growth in treated wells.The Gi₅₀ calculated for the compounds is the estimated concentration atwhich growth is inhibited by 50% compared to control, i.e., theconcentration at which:

100×[(Treated₄₈ −T ₀)/(Control₄₈ −T ₀)]=50.

[0184] All concentrations of compounds are tested in duplicate andcontrols are averaged over 12 wells. A very similar 96-well plate layoutand Gi₅₀ calculation scheme is used by the National Cancer Institute(see Monks, et al., J. Natl. Cancer Inst. 83:757-766 (1991)). However,the method by which the National Cancer Institute quantitates cellnumber does not use MTS, but instead employs alternative methods.

[0185] Calculation of IC₅₀:

[0186] Measurement of a composition's IC₅₀ for KSP activity uses anATPase assay. The following solutions are used: Solution 1 consists of 3mM phosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (SigmaA-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (Sigma T-7402), 10ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2consists of 1 mM NADH (Sigma A8129), 0.2 mg/ml BSA (Sigma A7906),pyruvate kinase 7 U/ml, L-lactate dehydrogenase 10 U/ml (Sigma PO294),100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (Sigma D9779),5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003-01), and 1 mMEGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions) of thecomposition are made in a 96-well microtiter plate (Corning Costar 3695)using Solution 1. Following serial dilution each well has 50 μl ofSolution 1. The reaction is started by adding 50 μl of solution 2 toeach well. This may be done with a multichannel pipettor either manuallyor with automated liquid handling devices. The microtiter plate is thentransferred to a microplate absorbance reader and multiple absorbancereadings at 340 nm are taken for each well in a kinetic mode. Theobserved rate of change, which is proportional to the ATPase rate, isthen plotted as a function of the compound concentration. For a standardIC₅₀ determination the data acquired is fit by the following fourparameter equation using a nonlinear fitting program (e.g., Grafit 4):$y = {\frac{Range}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + {Background}}$

[0187] where y is the observed rate and x the compound concentration.

[0188] The K_(i) for a compound is determined from the IC₅₀ based onthree assumptions. First, only one compound molecule binds to the enzymeand there is no cooperativity. Second, the concentrations of activeenzyme and the compound tested are known (i.e., there are no significantamounts of impurities or inactive forms in the preparations). Third, theenzymatic rate of the enzyme-inhibitor complex is zero. The rate (i.e.,compound concentration) data are fitted to the equation:$V = {V_{\max}{E_{0}\left\lbrack {I - \frac{\left( {E_{0} + I_{0} + {Kd}} \right) - \sqrt{\left( {E_{0} + I_{0} + {Kd}} \right)^{2} - {4E_{0}I_{0}}}}{2E_{0}}} \right\rbrack}}$

[0189] where V is the observed rate, V_(max) is the rate of the freeenzyme, I₀ is the inhibitor concentration, E₀ is the enzymeconcentration, and K_(d) is the dissociation constant of theenzyme-inhibitor complex.

[0190] Several representative compounds of the invention (as theirfumarate salts) were tested as described above and found to exhibit Ki'sless than 10 μM. Their structures are as shown:

[0191] The phenothiazine compounds inhibit growth in a variety of celllines, including cell lines (MCF-7/ADR-RES, HCTI 5) that expressP-glycoprotein (also known as Multi-drug Resistance, or MDR⁺), whichconveys resistance to other chemotherapeutic drugs, such as pacilitaxel.Therefore, the phenothiazines are anti-mitotics that inhibit cellproliferation, and are not subject to resistance by overexpression ofMDR⁺ by drug-resistant tumor lines.

[0192] Compounds of this class were found to inhibit cell proliferation,although GI₅₀ values varied. GI₅₀ values for the phenothiazine compoundstested ranged from 200 nM to greater than the highest concentrationtested. By this we mean that although most of the compounds thatinhibited KSP activity biochemically did inhibit cell proliferation, forsome, at the highest concentration tested (generally about 20 μM), cellgrowth was inhibited less than 50%. Many of the compounds have GI₅₀values less than 10 μM, and several have GI₅₀ values less than 1 μM.Anti-proliferative compounds that have been successfully applied in theclinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'sthat vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM,doxorubicin is 63 nM, 5-fluorouracil is 1 μM, and hydroxyurea is 500 μM(data provided by National Cancer Institute, Developmental TherapeuticProgram, http://dtp.nci.nih.gov/). Therefore, compounds that inhibitcellular proliferation at virtually any concentration may be useful.However, preferably, compounds will have GI₅₀ values of less than 1 mM.More preferably, compounds will have GI₅₀ values of less than 20 μM.Even more preferably, compounds will have GI₅₀ values of less than 10μM. Further reduction in GI₅₀ values may also be desirable, includingcompounds with GI₅₀ values of less than 1 μM.

We claim:
 1. A method of treating cellular proliferative diseasescomprising administering a compound of the formula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R⁴ and R⁵ are independently chosen fromhydrogen, alkyl, substituted alkyl, alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ and R⁴taken together with the intervening atoms form one or more five- toseven-membered rings, said ring optionally substituted with one or morealkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, with the proviso that said phenothiazine must contain atleast one additional five- to seven-membered ring, or a pharmaceuticallyacceptable salt thereof.
 2. A method of treating a disorder associatedwith KSP kinesin activity comprising administering a compound of formula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R⁴ and R⁵ are independently chosen fromhydrogen, alkyl, substituted alkyl, alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ and R⁴taken together with the intervening atoms form one or more five- toseven-membered rings, said ring optionally substituted with one or morealkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, with the proviso that said phenothiazine must contain atleast one additional five- to seven-membered ring, or a pharmaceuticallyacceptable salt thereof.
 3. A method of inhibiting KSP kinesincomprising contacting KSP kinesin with a compound of formula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R⁴ and R⁵ are independently chosen fromhydrogen, alkyl, substituted alkyl, alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ and R⁴taken together with the intervening atoms form one or more five- toseven-membered rings, said ring optionally substituted with one or morealkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, with the proviso that said phenothiazine must contain atleast one additional five- to seven-membered ring, or a pharmaceuticallyacceptable salt thereof.
 4. A method according to claim 1, 2 or 3wherein wherein R³ is hydrogen and R² and R⁴ form a five- toseven-membered ring.
 5. A method according to claim 4 wherein saidphenothiazine has the formula


6. A method according to claim 1, 2 or 3 wherein R² and R³ are hydrogenand R⁵ is alkylaryl or substituted alkylaryl.
 7. A method according toclaim 1 or 2 wherein said disease or disorder is chosen from the groupconsisting of cancer, hyperplasia, restenosis, cardiac hypertrophy,immune disorders and inflammation.
 8. A method according to claim 4wherein said disease or disorder is chosen from the group consisting ofcancer, hyperplasia, restenosis, cardiac hypertrophy, immune disordersand inflammation.
 9. A method according to claim 5 wherein said diseaseor disorder is chosen from the group consisting of cancer, hyperplasia,restenosis, cardiac hypertrophy, immune disorders and inflammation. 10.A method according to claim 6 wherein said disease or disorder is chosenfrom the group consisting of cancer, hyperplasia, restenosis, cardiachypertrophy, immune disorders and inflammation.
 11. A phenothiazine ofthe formula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R^(4a) is chosen from hydrogen and loweralkyl; and R^(5a) is chosen from alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ andR^(4a) taken together with the intervening atoms form one or morefive-to seven-membered rings, said ring optionally substituted with oneor more alkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, or a pharmaceutically acceptable salt thereof.
 12. Aphenothiazine according to claim 11 wherein R³ is hydrogen and R² andR^(4a) form a five- to seven-membered ring.
 13. A phenothiazineaccording to claim 12 of formula


14. A phenothiazine according to claim 13 wherein R^(5a) is benzyl orsubstituted benzyl.
 15. A phenothiazine according to claim 11 wherein R²and R³ are hydrogen and R^(5a) is alkylaryl or substituted alkylaryl.16. A phenothiazine according to claim 15 wherein R^(5a) is benzyl orsubstituted benzyl.
 17. A method of screening for KSP kinesin modulatorscomprising: combining a kinesin, a candidate bioactive agent and acompound of the formula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R⁴ and R⁵ are independently chosen fromhydrogen, alkyl, substituted alkyl, alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ and R⁴taken together with the intervening atoms form one or more five- toseven-membered rings, said ring optionally substituted with one or morealkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, with the proviso that said phenothiazine must contain atleast one additional five- to seven-membered ring, or a pharmaceuticallyacceptable salt thereof, and determining the effect of said candidatebioactive agent on the activity of said kinesin.
 18. A method ofscreening for compounds that bind to KSP kinesin comprising: combining akinesin, a candidate bioactive agent and a labeled compound of theformula

wherein R¹ is hydrogen, halogen or CF₃; R² is chosen from hydrogen andlower alkyl; R³ is hydrogen; R⁴ and R⁵ are independently chosen fromhydrogen, alkyl, substituted alkyl, alkylaryl, substituted alkylaryl,alkylheteroaryl and substituted alkylheteroaryl; or any of R², R³ and R⁴taken together with the intervening atoms form one or more five- toseven-membered rings, said ring optionally substituted with one or morealkyl, aryl, alkoxy, halo, alkylaryl or substituted alkylarylsubstituents, with the proviso that said phenothiazine must contain atleast one additional five- to seven-membered ring, or a pharmaceuticallyacceptable salt thereof; and determining the binding of said candidatebioactive agent to said kinesin.